Many agriculturally important crops are susceptible to infection by plant viruses. These viruses can seriously damage a crop and drastically reduce its economic value to the grower. This eventually leads to a higher cost for the consumer. Attempts to control or prevent infection of a crop by a plant virus have been made, yet viral pathogens continue to be a significant problem in agriculture. For instance, many economically important viruses are spread by insects. The insecticides are used to kill these insects in order to prevent crop plant infection and spread of the virus. However, insecticide application is expensive and can kill non-target organisms and posses significant health risks to humans.
Scientists have recently developed means to produce virus resistant plants using genetic engineering techniques. Such an approach is advantageous, in that the means for providing the protection is incorporated into the plant itself and is passed on to its progeny. A host plant is resistant if it possesses the ability to prevent infection, to suppress or retard the multiplication of a virus, and to suppress or retard the development of pathogenic symptoms. "Resistant" is the opposite of "susceptible," and definitions of the terms are described in Cooper and Jones, 1983. Several different types of host resistance to viruses are recognized. The host may be resistant to: (1) establishment of infection, (2) virus multiplication, or (3) viral movement.
Genes which interfere with the process of virus replication and/or infection may be expressed in transgenic plants to protect against viral disease. It has previously been shown that expression of a plant virus capsid protein, which is termed the coat protein (CP), in a plant can confer resistance to the homologous virus and to related viruses (Abel et. al., 1986; Tumer et. al., 1987: Cuozzo et al., 1988; Hemenway et. al., 1988; Stark et. al., 1989; Lawson et. al. 1990; Kaniewski et. al., 1990). In these studies, resistance to virus disease is defined as either reduced incidence of infection, delayed symptom development, reduced virus replication or viral antigen levels, or slower to no systemic virus movement. Expression of the virus coat protein in these transgenic plants may be responsible for the observed effects in the reduction of virus disease by an as yet undetermined mechanism (Abel et. al.,1986; van Dun et. al., 1988 -A). This type of protection against viral infection is termed coat protein-mediated resistance.
Even though coat protein-mediated viral resistance has proven to be useful in a variety of situations, it may not always be the most effective means for providing viral resistance. In such instances, other methods maybe useful for conferring viral resistance to plants. Other technologies have been demonstrated or proposed which affect virus or disease development. Examples of these are: antisense coat protein (Cuzzo et. al., 1988), satellite RNA (Harrison et. al., 1987), ribozymes (Walbot et. al., 1988), defective interfering molecules (Morch 1987), viral nonstructural genes (Golemboski et. al., 1990),(Braun et al., 1992) antibodies (Hiatt, 1990), PR proteins (Bol et. al., 1990) and antiviral proteins (Irvin et. al., 1980).
A fragment of the putative replicase gene from tobacco mosaic virus (TMV), a viral nonstructural gene, recently has been found to provide resistance against TMV when expressed in tobacco plants (Golemboski et. al., 1990). In TMV, two proteins, the 183 kilodalton (kDa) and 126 kDa proteins, have been speculated to be replicase components, as the expression of both proteins are necessary for normal multiplication in tobacco plants (Ishikawa et. al., 1986). In addition, these two proteins contain evolutionarily conserved motifs, such as the NTP binding motif and the GDD domain, that are often found in other known RNA dependent RNA polymerases or replicase genes (Koonin, 1991). The NTP binding motif is the amino acid sequence G-X-X-X-X-G-K-X' where G is glycine, X is any amino acid, and X' is usually a serine (S) or threonine (T). (Gorbalenya et. al., 1988). The GDD motif is a larger domain, which is characterized by the presence of three invariant residues: glycine (G), followed by two aspartic acid (D) residues. GDD domains are often found in replicase proteins and are believed to be involved in catalytic function (Hodgman, 1988). The 183 kDa protein is produced by a translational read-through of the 126 kDa termination codon (TAG). The 126 kDa protein contains the NTP binding motif. The 183 kDa protein contains both the NTP and GDD motifs. The region of the TMV genome that conferred resistance was the read-through portion of the 183 kDa putative replicase gene. This read-through portion has the coding capacity for a 54 kDa protein. The GDD domain is located within the 54 kDa and 183 kDa protein sequence. For a number of years, it has been speculated that the 54 kDa protein is made as a separate gene product. To determine the function of this putative gene, Golemboski et. al. (1990) transformed tobacco with this sequence, and unexpectedly found that the transgenic plants were resistant to TMV. Plants transformed with the gene encoding the 126 kDa protein were unprotected. No data were reported on the 183 kDa read-through protein.
The mechanism of the observed resistance by expressing a truncated form of the replicase (the GDD domain) is unclear. It has recently been demonstrated in the case of the TMV 54 kDa read-through protein (GDD domain) that expression of the protein is required for the observed resistance (Carr et. al., 1992.).
Others have conducted protection experiments with transgenic plants expressing components of non-structural viral proteins. For Example, van Dun et. al. (1988) analyzed protection in tobacco plants expressing either of two genes encoding proteins involved in the replication of alfalfa mosaic virus (AlMV). These plants were transformed with cDNA's for RNAs 1 or 2 of AlMV, which encode proteins P1 and P2, respectively. The polypeptides, P1 and P2, encoded by these RNAs have amino acid similarities to other viral replicases, and both RNAs are known to be essential for replication. The NTP and GDD motifs for AlMV reside on different RNAs and consequently different proteins. Specifically, P1 contains the NTP binding motif and P2 contains the GDD motif. Plants expressing either RNA1 or RNA2 were unprotected against infection by AlMV. In addition, plants expressing both RNAs 1 and 2 were also unprotected against infection by AlMV (Taschner et. al., 1991).
Buck et. al. (PCT publication WO 92/03539) have described the use of various techniques to prevent the expression or function of a cucumber mosaic viral replicase in order to provide viral resistance in plants. The techniques employed or disclosed in this publication to accomplish resistance included: (1) antisense technology (wherein a complementary RNA to that coding for the full length replicase can be expressed); (2) expression of a gene coding for the production of an antibody specific for one of the three virally-encoded components of the replicase (viral encoded polypeptides P1a and P2a, and polypeptide P50 from tobacco); and (3) expression of a ribozyme specific for the RNA coding for one of the components of the replicase.
Potato leafroll virus (PLRV) and barley yellow dwarf virus (BYDV) are members of the luteovirus plant virus group. Both are positive-sense, single-stranded RNA viruses. To form a viral particle, the viral RNA is encapsidated by the coat protein to give the characteristic isometric shape typical of viruses in the luteovirus group. BYDV is transmitted to cereal crops and wild grass species by aphids.
Other members of the luteovirus group are: bean leaf roll (BLRV), beet western yellow virus (BWYV), carrot red leaf virus (CtRLV), groundnut rosette assistor virus (GRAV), Indonesian soybean dwarf virus (ISDV), soybean dwarf virus (SDV), and tobacco necrotic dwarf virus (TNDV) and Solanum yellows virus (SYV). Other possible members are beet yellow net virus, celery yellow spot virus, chickpea stunt virus, cotton anthocyanosis virus, filaree red leaf virus, grapevine ajinashika virus, milk vetch dwarf virus, millet red leaf virus, Physalis mild chlorosis virus, Physalis vein blotch virus, raspberry leaf curl virus, tobacco distorting virus, tobacco yellow net virus, and tobacco yellow vein assistor virus.
The luteovirus group can be divided into two subgroups of viruses which contain many strains. The most economically significant members of subgroup I are BYDV-MAV, BYDV-PAV, and BYDV-SGV. The most economically significant members of subgroup II are BYDV-RGV, BYDV-RMV, BYDV RPV, PLRV and BWYV. These two subgroups differ principally in aspects of their genome organization. The genome organization of BYDV subgroup I and BYDV subgroup II is illustrated in FIG. 13 and reviewed by Matthews (1991) and Murphy (1995). The genome organization of BYDV subgroup I strains includes an open reading frame (ORF) 1 which translates into a 39 kDa protein and an ORF2 which encodes a 60 kDa protein, however these two ORFs are translated by a frameshift of the reading frame near the 3' terminus of ORF1 to produce a 99 kDa protein. This 99 kDa protein is required for replication (Mohan et. al., 1995). There is no evidence that the 60 kDa ORF is translated independent of the frameshift mechanism. Therefore the gene for the full length BYDV subgroup I replicase includes the portion of the viral genome which contains ORF1 and ORF2. The amino acid sequence identity comparisons for ORF1 and ORF2 of BYDV-PAV and BYDV-MAV is 98% and 96% respectively (Ueng, et. al., 1992). The genome of BYDV subgroup II viruses also contain 2 ORFs which overlap and which when a translational frameshift occurs will translate a full length replicase gene.
The host range of BYDV is limited to the Gramineae, which includes crops such as barley, oats, wheat, rice, corn, rye and wild and cultivated forage grasses. BYDV is transmitted in a persistent manner by aphids. Early season infection can result in substantial crop losses. An estimated 2-5% of the worlds wheat crop is lost to BYDV caused disease every year. The current management practices primarily involve the use of contact and systemic insecticides. There is currently no effective genetic resistance in wheat to BYDV. Thus, it would be a significant contribution to the art to develop an alternative method to those currently available that is effective for conferring BYDV resistance in wheat and other Poaceae crops.
PLRV RNA possesses a genome-linked proteinaceous unit at the 5' terminus and the 3' end does not contain a poly A tail. (Mayo et. al., 1982). The PLRV genomic RNA replicates through RNA intermediates in a DNA-independent fashion. PLRV RNA has six open readings frames (ORFs) (FIG. 1). The organization of the PLRV genome is reviewed in Martin et. al. (1990). In the 5' half of genomic RNA, a small ORF (ORF1) which encodes a 28 kDa protein is followed by two large ORFs (ORF 2a and ORF 2b), which may code for a 70 kDa and a 67 kDa protein, respectively. ORF2a and ORF2b is proposed to encode a putative replicase protein by virtue of its sequence similarity to other known replicase genes. In particular, ORF2a and ORF2b contain the motifs characteristic of NTP domain (Habili et. al., 1989) and RNA polymerases (Kamer et. al., 1984). ORF2b contains the GDD motif often found in replicase proteins and is believed to be involved in catalytic function. Henceforth we refer to the PLRV open reading frames 2a and 2b as the putative replicase or replicase. In PLRV isolate LR-7 Washington, ORF 2a and ORF 2b overlap by 579 nucleotides. Because ORF 2b lacks an AUG translational start codon in this region, it is postulated that ORF 2b is expressed by ribosomal frameshifting of ORF 2a (Mayo et. al., 1989).
A 2.3 kb subgenomic RNA transcribed from the minus strand message as part of the normal infection cycle is responsible for the translation of ORF3 (the coat protein (CP) gene), ORF4 (17 kDa putative nucleic acid binding protein (Tacke et. al., 1991) and ORF5 (56 kDa read-through protein, Bahner et. al., 1990). The CP gene is separated from a 56 kDa ORF by an amber stop codon (TAG). There is evidence that the 56 kDa protein is translated by suppression of the CP gene amber stop codon (Bahner et. al., 1990). Thus the 56 kDa ORF is expressed as a read-through product in a similar manner as the TMV 183 kDa protein.
The host range of PLRV is limited to members of the Solanaceae family of which potato, tobacco, tomato and peppers are important members. Commercially important potato cultivars to which the present invention may be applied include but are not limited to: Russet Burbank, Shepody, Atlantic, Norchip, and Superior.
PLRV is transmitted in a persistent manner by aphids. One of the most serious viral problems in the potato industry is infection of the potato crop with potato leafroll virus (PLRV). Infection of potato by PLRV causes a reduction in both quality and yield of the potato crop, thus resulting in substantial economic losses. In Russet Burbank potato, the tuber symptom of PLRV infection is a phloem necrosis called "net necrosis". The virus induces a necrosis of the phloem as a primary pathological change (Shepardson et. al., 1980). This necrosis affects the processing quality of the potato and economically impacts the potato grower by reducing the value of the crop. Economic losses caused as a result of PLRV infection have been estimated to be approximately 5% of the world potato crop. Current management of PLRV infection of a crop involves the use of insecticides to control the aphids that transmit the virus, but this method of control is expensive, potentially dangerous to the environment, and not totally effective.
The host range of other luteoviruses may be more extensive. For example, the host range of beet western yellow virus includes 23 dicotyledonous families, and may affect the following crops: sugar beet, table beet, spinach, lettuce, soybean, broccoli, cauliflower, radish, turnip, pea, broad bean, chickpea, flax, sunflower, mustard, clover, cabbage, swede, rape, crambe, pepper, pumpkin, watermelon, cucumber,tomato, etc.
As can be seen from the foregoing discussion, barley yellow dwarf and potato leafroll virus infection of various plants is a serious problem encountered in agriculture today. Thus, it would be a significant contribution to the art to develop an alternative method to those currently available that is effective for conferring viral resistance to plants.